The plasma membrane and the underlying cortical cytoskeleton undergo continuous dynamic interplay that is necessary for many essential cellular processes, including signal transduction, migration, endo-/exo-cytosis, and inter- and intra-cellular attachments/adhesion. Thus, the proteins that regulate these interactions and their function are of general importance in biology. The Wiskott Aldrich Syndrome (WAS) protein family, which act as effectors of Rho family GTPases, polymerize actin through the Arp2/3 complex. The two founding WAS family members, WASP and SCAR/WAVE, are involved in membrane-cytoskeletal interactions important for processes including membrane/vesicle trafficking, morphogenesis, angiogenesis, inflammatory immune response, and when mis-regulated, in pathogen infection, bleeding/bruising, immune deficiency and cancer metastasis. We have recently identified a third subfamily of WAS family proteins, designated WASH, that is conserved from Entamoeba to humans. The long-term goal of this proposal is to delineate both the cytoplasmic and nuclear function(s) of WASH and the developmental consequences of these processes going awry. The specific aims of this proposal are 1) to determine the role(s) of WASH in the regulation of actin and microtubule cytoskeletal coordination and cytoskeletal-membrane crosstalk using genetic, developmental, molecular/biochemical, and cell biological approaches;2) to delineate the mechanisms of WASH function in hemocyte developmental and chemotactic migrations;and 3) to investigate the effect of WASH on nuclear architecture and organization using a combination of comparative bioinformatic, cell biological, and high-resolution microscopic approaches. The wealth of techniques, sophisticated live imaging, and reagents available in Drosophila make it an excellent, genetically manipulable, organism for studying the functions of WASH in vivo. Results obtained from these projects are expected to have wide implications as WASH is conserved throughout organisms. WASH will also serve as a reference protein for revealing general organizational principles and/or mechanisms for the roles of other actin-binding and cytoplasmic (cytoskeletal) proteins/families and will provide new insight into mechanisms controlling universal molecular and cell biological events both in and out of the nucleus. The information gathered in these projects will be important for understanding how WASH functions to control key cellular processes in normal development or when mis-regulated in disease states, and in the longer term, for the potential development of novel anti- disease drugs and/or therapies, or to enhance the effectiveness of existing treatments. PUBLIC HEALTH RELEVANCE: Wiskott-Aldrich Syndrome (WAS) is an X-linked genetically transmitted disease that presents clinically with symptoms ranging from mild eczema to severe bruising/bleeding, immune deficiency, and cancer. The proposed studies will examine the functions of a newly identified subfamily of WAS family proteins, named WASH, which functions to reorganize cytoskeleton and membrane structures in cells in response to various signals, thereby causing cells to change shape and/or move. As WASH is highly conserved, information learned from Drosophila can be extrapolated to higher organisms, including humans. These studies are expected provide new information on its role(s) not only in normal processes, but also in disease conditions and cancer onset/progression, and may provide a basis for new avenues of therapeutic development.